Defects in DNA repair enzymes contribute to tumorigenesis. Nucleases cleave the damaged DNA fragments induced by radiation and other environmental insults. They play an important role in the multiple enzyme processes of DNA replication, repair, and DNA recombination. Most nucleases in major well-defined repair pathways such as nucleotide. excision repair and mismatch repair pathways require DNA damage recognition proteins that interact with them before they act. Flap endonucleases (FEN), in contrast, can recognize the damaged DNA fragments and cleave them without accessory proteins. This property may suggests a unique structural basis and in turn a different mechanism for the reaction compared to other DNA repair enzymes. With this unique property, its roles in DNA repair and replication, and the relatively small size of the protein, human flap endonuclease-1 (hFEN-1) is a very attractive enzyme for the structural and functional analysis. Three functional domains in human FEN-1 protein have been proposed. They may function in DNA substrate binding, catalysis, protein-protein interaction and nuclear localization. The proposed research is designed to establish a detailed structural understanding of the catalytic center for the DNA substrate recognition, binding, and cleavage; to analyze the structural elements responsible for protein-protein interaction with proliferating cell nuclear antigen; and to map the nuclear localization signal in FEN-1. We will perform site-directed mutagenesis in each of the functional domains of FEN-1 and characterize mutant proteins using kinetic, biochemical assays, 3-D structure comparative analysis, and green fluorescence protein-driven microscopy. The information available from this systematic structural and functional analysis of the FEN-1 enzyme will be very useful in elucidating the molecular mechanisms of structure-specific endonucleases in DNA repair and replication, and in identifying of critical amino acids in mutator genes when clinically screening cancers.